Unusual pulmonary thromboembolism in a Dirofilaria immitis infected dog Tromboembolismo

The nematode Dirofilaria immitis and its endosymbiont Wolbachia are the agents of canine heartworm (HW) disease. The worm´s main habitats are the pulmonary arteries, which elicit inflammation and disorders of the coagulation process. Clinical signs vary according to multiple factors, including worm burden, individual reaction, and duration of infection. This report describes an unusual case of pulmonary thromboembolism secondary to HW infection. A 7-year-old female dachshund presented with a history of cough, dyspnea, and two syncope episodes. On physical examination, the oral mucosa was slightly discolored, the femoral pulse was weak, respiratory distress was present, and the abdomen distended. Echocardiography showed severe right atrioventricular and pulmonary artery enlargement and a large structure with high echogenicity in the artery. After the dog died naturally, the lungs and heart were examined. Only one worm was found in the right ventricle, and a large thrombus was found in the main pulmonary artery. Histologically, fibrin thrombi and fragments of parasites were present inside the pulmonary artery branches. These findings highlight the importance of a thorough clinical evaluation of HW patients and confirm that only a few worms can threaten an animal’s life. Therefore, veterinarians must enforce chemoprophylaxis once treatment is life threatening. à avaliação macroscópica e microscópica. Apenas um grande foi encontrado na artéria pulmonar Histologicamente, D.


Introduction
Dirofilaria immitis is a nematode that inhabits the pulmonary vessels and right chambers of the heart that commonly affects dogs through transmission by their vector mosquitoes (Rawlings, 1986). This parasite and its endosymbiont Wolbachia are the agents of canine heartworm (HW) disease, which can present asymptomatic to a severe form of the disease depending on the parasitic burden, duration of infection, and individual immunological reactions in the face of the infection (Bowman & Atkins, 2009;Dillon et al., 1995). Disease tends to be more severe in long-term infections with heavy worm burden, and clinical signs may include cough, dyspnea, intolerance to exercise, syncope, ascites, and even death (Rawlings, 1986;McCall et al., 2008;Labarthe et al., 2009;Mircean et al., 2017).
The occurrence of pulmonary thromboembolism (PTE), due to the presence of adult worms, can obliterate the passage of blood from the heart to the lungs and is well known and described, although rare (McHaffie, 2012). The presence of PTE is a life-threatening event due to the possibility of completely obstructing the blood flow to the lungs, although the obstruction can be partial, leading to Cor pulmonale (MacNee, 1994;Haddad et al., 2008;McCall et al., 2008).
Although the presence of the parasite and its endosymbiont, Wolbachia, elicits PTE through physical mechanisms and direct inflammation (Rawlings, 1986;Calvert & McCall, 1998), indirect factors may also contribute to its development. These factors include platelet function activation, fibrinolytic activators, and factor Xa, which are common during heartworm infection (González-Miguel et al. 2015;Takashima et al., 2017;Diosdado et al., 2020). The aim of this report was to describe an unusual case of pulmonary thromboembolism secondary to D. immitis infection.

Case description
A 7-year-old dachshund spayed bitch weighing 9kg presented with cough and dyspnea for the past seven days. She presented with two syncope episodes during this week. The owner reported that the animal was under alternative treatment for HW disease based on doxycycline and moxidectin. On physical examination, the oral mucosa was slightly discolored, the femoral pulse was weak, respiratory distress was present, and the abdomen distended. Cardiac auscultation revealed the presence of systolic heart murmur grade III/VI at the tricuspid valve area, and diastolic murmur at the pulmonary valve area grade III/VI. Pulmonary auscultation crackle sounds were mainly heard in the right lung. Based on the critical clinical conditions, the patient was submitted to oxygen therapy and additional examinations to determine the main cause of the signs.
Abdominal ultrasound, echodopplercardiography, and electrocardiogram (EKG) were performed immediately. Ultrasound showed hepatomegaly, dilation of the liver's blood vessels, and a mild volume of abdominal fluid. Echocardiography in B-mode showed severe right atrioventricular dilatation, slight hypertrophy of the right ventricular wall and interventricular septum, reduction in the left ventricular lumen, and enlargement of the pulmonary artery. During the examination of the main pulmonary artery, a large structure with high echogenicity was observed. The evaluation with color-flow Doppler demonstrated the presence of severe tricuspid regurgitation ( Figure 1) and moderate pulmonary regurgitation ( Figure 2). The evaluation using continuous-wave Doppler showed that tricuspid regurgitation had a maximum peak velocity of 4.48 m/s.

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Unusual pulmonary thromboembolism in a Dirofilaria immitis infected dog → shows moderate pulmonary regurgitation. The EKG (DII lead; N; 50 mm/s) showed the presence of a p-pulmonale wave (0.5mV), the presence of an elevated ST segment (0.4 mV), an increase in the T wave amplitude (25% greater than the R wave),the presence of a deep S wave (-2.2 mV), right cardiac axis deviation (between -90º and -120º), and an average heart rate of 150 bpm ( Figure 3).  Before chest radiography and therapeutic approach could be performed, the patient died due to cardiorespiratory arrest.
After the owner's consent for postmortem examination of the thorax, the thoracic cavity was opened and examined. Only two female worms were found. One worm was found free in the cavity (Figure 4), and the other was found entangled at the trabeculae carneae ( Figure 5) of the right ventricle. The right chambers of the heart were enlarged, and red-blue areas were observed in the right lobes (superior, middle, and inferior) of the lung (Figure 6). A thrombus of approximately 5cm in length was found in the main pulmonary artery (Figure 7). Lung samples were individually fixed in 10% formaldehyde and sent for histopathological evaluation.
Histologically, the lung parenchyma showed areas of atelectasis contiguous to compensatory emphysema and diffuse thickening of alveolar walls with the presence of macrophages also observed inside the alveolar cavity. Thickening of the artery wall and intima with villous projections was observed in the main pulmonary artery and its branches inside the parenchyma (Figures 8, 9). Necrotic areas surrounded by inflammatory cells and fibroblasts were also observed. Fibrin thrombi, some already organized and recanalized, were present inside pulmonary artery branches in the parenchyma. Fragments of parasites inside the artery branches ( Figure 10) and a large fibrin thrombus was seen in the main pulmonary artery (Figure 11).

Discussion
Although the patient presented with clinical signs compatible with HW infection and with the previous treatment the dog was receiving, further investigation was needed to exclude other causes of heart or lung diseases (American Heartworm Society, 2020) and to evaluate the extension of the lesions.
Physical examination showed signs of decreased cardiac output (discolored oral mucosa, weak femoral pulse, and distended abdomen), suggesting that the pathological process was compromising heart function. However, it was necessary to perform the requested exams to ensure the extent of the lesions and to lead treatment (Gompf, 2008;American Heartworm Society, 2020). Lung sounds detected, despite classically indicating cardiogenic pulmonary edema, when evaluated with the other physical examination findings (abdominal distention, murmur in the pulmonary and tricuspid areas) were not consistent. These findings suggested the inflammatory onset in the lungs, due to HW disease (Atwell, 1988;Ames & Atkins, 2020), since the patient was known to be HW-infected.
Echocardiographic evaluation suggested pulmonary hypertension as a consequence of the thrombus. This was confirmed by the absence of morphological abnormalities at the tricuspid and pulmonary valve apparatus associated with increased tricuspid regurgitation velocity, enlargement of the main pulmonary artery, and the presented clinical signs (Ames & Atkins, 2020;Reinero et al., 2020). The severity of the case was further reiterated by the presence of a ST-segment elevation on the EKG, an uncommon finding in dogs that reflects the low supply of oxygen to the cardiac muscle and may indicate rare canine myocardial ischemia (Santilli et al., 2020).
It is well known that HW disease therapy can present systemic complications. When worms die, there is intense inflammatory stimulus due to the worm's and Wolbachia´s antigens that trigger pulmonary reactions and can even elicit verminotic embolism (Rawlings, 1986;Turner et al., 2009;Kramer & Genchi, 2014). However, this correlation cannot be either affirmed or excluded in the present case since the animal´s history is veiled. Therefore, to increase the capacity to choose the best adulticidal treatment regimen to reduce the risk for the patient, a complete assessment of the patient's health condition must be performed. A thorough clinical evaluation must always be performed, including blood count, biochemistry, and radiography. Echocardiography may be also needed (American Heartworm Society, 2020).Although worm burden is directly related to the clinical manifestations (Dillon et al., 1995;Bowman & Atkins, 2009), in the present case only one adult worm was found inside the heart, in addition to the low number of parasite fragments found inside the large fibrin thrombus, suggesting that the severity of the disease was due to the patient's inflammatory reaction to injury (Ames & Atkins, 2020), more than to the parasite burden. In association with the main lesions, thickening of the alveolar wall must have reduced alveolar complacency, resulting in emphysema (Sutton& Atwell, 1985), reducing the air passage leading to atelectasis (West, 2014).
The presence of worms and their endosymbionts can cause coagulation disorders that are associated with endothelial injury, favoring thrombus formation (González-Miguel et al., 2015;Takashima et al., 2017;Diosdado et al., 2020). As there are no hematological work results available, it is impossible to include or exclude coagulation disorder as the primary cause of thrombi formation. Nevertheless, the number of thrombi found in the pulmonary arteries, the rapid onset of disease, and clinical worsening of the patient's health strongly suggest that coagulation disorders play an important role.
The presence of an inflammatory process is expected because the presence of the worms and its endosymbiont trigger the host's immune system and predispose thromboembolic events (Rawlings, 1986;Carretón et al., 2013;Yoon et al., 2017). Small fragments of the thrombus may have got detached and reached the pulmonary artery branches as identified by microscopy. This process presumptuously was the main cause of tissue ischemia, translated in the form of necrotic areas (Shapiro, 2009) as identified. The presence of recanalization reflects the attempt of reperfusion of the lung in the face of ischemia, suggesting that the obstruction of these vessels preceded the total obstruction of the main pulmonary artery (Wagenvoort, 1995).
The presence of parasite fragments inside the thrombus suggests the presence of at least a third parasite. It is possible to infer that, due to its location, its presence was a determinant of thrombus formation. The presence of the endothelial lesion observed, associated with platelet activation and reduction of thrombolytic mechanisms expected in HW infections may have contributed to thrombogenesis (González-Miguel et al., 2015;Takashima et al., 2017;Falcón-Cordón, et al, 2019;Diosdado et al., 2020).

Conclusions
These findings highlight the importance of a thorough clinical evaluation of HW-infected patients before any adulticidal treatment. This case confirms that only few worms are enough to threaten an animal's life. Therefore, small animal practitioners must always enforce chemoprophylaxis to avoid HW infection; once treatment, even in the face of a small worm burden, is life threatening.

acknowledgements
The authors thank Maria Isabel Rossi for the consulting in the field of pathology and Celeste da Silva Freitas de Souza and Natalia Freitas de Souza for performing the photomicrographies.